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Biomedical subjects

B Lambert

Publications and source records attributed to B Lambert.

At least 91 records · Page 5Linked to original sources
PubMed1990

Recognition by the DNA repair system of DNA structural alterations induced by reversible drug-DNA interactions.

Ditercalinium (NSC 335153) was synthesized as a bifunctional DNA intercalator. It is made of two 7-H pyridocarbazole rings joined by a rigid bis-ethyl bispiperidine chain. It binds to DNA with high affinity and elicits anti-tumor activity on a variety of animal tumors. 1H n.m.r. studies of ditercalinium bis-intercalated into d(CpGpCpG)2 have shown that the intercalation process occurs from the large groove of the DNA helix while the two intercalated rings are separated by two base pairs. Because of the linking chain rigidity of ditercalinium, DNA conformation has to be altered to permit the intercalation of the two rings. DNA must be bent toward the minor groove. In E. coli, ditercalinium elicits a specific toxicity on polA strains which is suppressed by an additional uvrA mutation. In vitro, the purified UvrA and UvrB proteins bind to the DNA-ditercalinium complex in an ATP dependent manner. The UvrABC complex induces single-strand nicks, but only when ditercalinium is bound to negatively supercoiled DNA. The life-time of the UvrAB-DNA-ditercalinium complex is greater than 50 min when free ditercalinium concentration is maintained constant in the incubation medium. The cytotoxicity of ditercalinium in E. coli results from the induction of a futile and abortive DNA repair. The reversible ditercalinium-DNA complex mimics a bulky DNA lesion, yet the UvrABC endonuclease is unable to cope with a reversible lesion since it cannot eliminate the causative agent. The interaction of UvrA and UvrB proteins has also been studied with DNA and other DNA-binding drugs forming high-affinity complexes such as distamycin. The Uvr protein recognition process appears to be associated with specific DNA structural alterations. In eukaryotic cells, ditercalinium is concentrated in mitochondria. Mitochondrial DNA is rapidly and totally degraded. Mitochondrial DNA coded proteins being no longer synthesized, the respiratory chain is progressively inactivated. The stimulation of the glycolytic pathway allows the cells to continue growth for several generations. Dihydro-orotate dehydrogenase is located in the inner membrane of mitochondria and its activity is dependent on mitochondria energization. It becomes inactive after ditercalinium treatment. A drop of the pyrimidine pool is then observed. Complementation of treated cells with uridine decreases 10-fold the ditercalinium toxicity. The cellular delayed toxicity of ditercalinium results from the slow induction of a pyrimidineless state associated with the progressive inactivation of mitochondria. The results show that DNA structural alterations induced by reversible drug-DNA complexes can be recognized by DNA repair enzymes.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1990

An exonic point mutation of the androgen receptor gene in a family with complete androgen insensitivity.

We have discovered in the X-linked androgen receptor gene a single exonic nucleotide substitution that causes complete androgen insensitivity (resistance) in a sibship with three affected individuals. The mutation, a guanine-to-adenine transition, occurs at nucleotide number 2682 and changes the sense of codon 717 from tryptophan to a translation stop signal. Codon 717 is in exon 4, so the mutation predicts the synthesis of a truncated receptor that lacks most of its androgen-binding domain. The substitution abolishes a recognition sequence for the restriction endonuclease HaeIII. Amplification of exon 4 by the polymerase chain reaction followed by double digestion with HinfI and HaeIII permits facile recognition of hemizygotes and heterozygous carriers of the mutation.

Amino Acid Sequence↗
PubMed1989

Lung ventilation and perfusion scintigraphy in the follow up of repaired congenital diaphragmatic hernia.

To evaluate the effects of congenital diaphragmatic hernia (CDH) and pulmonary hypoplasia on subsequent lung function and development, we performed lung 133Xe ventilation and 99mTc perfusion scintigraphies in a group of infants who had undergone surgical repair of a severe left CDH with respiratory distress within the first 6 h of life. The initial lung scans performed in 15 children, 2-3 months of age, demonstrated a decreased ventilation in 7. In 9 children there was a trapping of 133Xe at the left lung base. Perfusion to the hernia side was reduced in 8 of the children. We re-evaluated 11 of these 15 patients after 1-2 years. The ventilation to the left lung was still decreased in 3, but perfusion remained decreased in 9. After 5 years, ventilation to the hernia side was normal in 4 of the 5 patients studied, whereas pulmonary blood flow was abnormal in 4. These results show a progressive improvement of ventilation with a persisting reduction of perfusion to the lung of the hernia side, suggesting a primary vascular pulmonary hypoplasia in CDH.

Female↗
PubMed1989

Induced recombination between duplicated neo genes stably integrated in the genome of CHO cells.

Homologous recombination between 2 truncated neo genes stably integrated in the genome of Chinese hamster ovary (CHO) cells was studied. A vector containing a functional gpt gene and 2 tandemly arranged G418 resistance (neo) gene fragments with about 400 bp of sequence homology was transfected into CHO cells. Clonal cell lines were established from transfected cultures and the spontaneous frequency of G418-resistant revertants was found to range between 1 x 10(-4) and 5 x 10(-4). The ability of the alkylating agents MMS and HN2 to induce recombination of the transfected neo genes was studied in 2 of the cell lines. After treatment with MMS at doses that reduced survival to 10% of the control these cell lines showed a dose-dependent increase in the frequency of G418-resistant revertants. No effect was observed after treatment with HN2. All G418-resistant subclones contained a new restriction fragment indicating that a whole neo gene had been formed by rearrangement in pairs of truncated neo genes. Hence, this system can be used to study molecular mechanisms and chemical inducibility of homologous recombination in mammalian cells.

Animals↗
PubMed1989

Hprt mutations and karyotype abnormalities in T-cell clones from healthy subjects and melphalan-treated ovarian carcinoma patients.

In vivo mutations at the locus for hypoxanthine phosphoribosyl transferase (hprt) were studied in 6-thioguanine (TG)-resistant T-lymphocyte clones from healthy male and female subjects and ovarian carcinoma patients treated with melphalan. Southern blot analysis of 108 clones showed alterations in 14% (4/29) of the clones from healthy males, 4.3% (2/47) of the clones from healthy females and 3.1% (1/32) of the clones from melphalan-treated patients. 2 of the 7 abnormal clones had a total deletion of the hprt gene; the others had partial deletions. Karyotype analysis of 82 clones revealed 1 clonal abnormality in 29 mutant clones from healthy males (3.6%). Loss or structural aberration of 1 X-chromosome occurred in 6% of the clones from healthy females. The frequency of karyotypic abnormalities (excluding those affecting one of the X-chromosomes) was significantly higher in clones from patients (37%) as compared to healthy females (5.9%). No aberration was found to affect the hprt locus at Xq27 in any of the 82 clones studied.

Blotting, Southern↗
PubMed1989

Mechanisms for recombination between stably integrated vector sequences in CHO cells.

Possible mechanisms for homologous recombination in CHO cells have been investigated using a stably integrated vector, pIII-14gpt. The vector contains 2 inactive neo gene fragments in tandem arrangement. Functional neo gene activity can be restored by recombination between homologous regions in the 2 fragments. Cells in which this event has taken place become resistant to the antibiotic G418. Possible mechanisms for neo gene reactivation in this system are unequal exchange between chromatids, intrachromatidal deletion and gene conversion. DNA from a total of 74 G418-resistant cell clones have been isolated, and analyzed on Southern blots using neo-specific probes. Rearrangements of neo-specific restriction fragments were found to have occurred in all cell clones. In 50% of the revertants, these rearrangements can be explained by a deletion which brings the complementary regions in the 2 neo gene fragments together. One single revertant (1.3%) shows a possible gene conversion event. The other isolated revertants (about 48%) contain more complex rearrangements. These results indicate that the predominating recombination mechanism for reactivation of the neo gene in this system is either a deletion within a chromatid or an unequal exchange between sister chromatids.

Animals↗
PubMed1989

Mutagen-induced recombination between stably integrated neo gene fragments in CHO and EM9 cells.

The ability of mutagenic agents to induce homologous recombination was studied in a 'normal' Chinese hamster ovary (CHO) cell clone (CHO:5) and a cell clone (EM9:2) derived from the presumptive DNA repair-deficient mutant cell line EM9, which has a high spontaneous sister-chromatid exchange (SCE) level and shows hypersensitivity towards monofunctional alkylating agents and bromodeoxyuridine (BrdUrd). The 2 cell clones have been transfected with and allowed to incorporate stable genomic inserts of the vector pIII-14gpt, which contains 2 tandemly arranged neo gene fragments with a common 400-bp region of homology. Recombination between the truncated neo genes gives rise to geneticin sulfate (G 418)-resistant revertants with a spontaneous frequency of about 10(-4) in both cell clones. In CHO:5 an increased frequency of revertants was obtained after treatment with methyl methanesulfonate (MMS) and mitomycin C (MMC), while HN2, benz[a]pyrene diolepoxide (BPDE) and X-irradiation gave negative results. EM9:2 showed about the same increase of revertants after treatment with MMS as CHO:5, but in a 10-fold lower dose range. HN2 as well as BrdUrd induced revertants in EM9:2. These results show that mutagenic agents (MMS, MMC, HN2, BrdUrd) can induce homologous recombination in this system. This effect does not seem to be an unspecific effect of DNA damage (no effect of X-ray and BPDE), or related to SCE induction in general (similar spontaneous and MMS-induced frequencies of revertants in CHO:5 and EM9:2). However, the positive effect of BrdUrd in EM9:2 and the difference between CHO:5 (negative) and EM9:2 (positive) with regard to HN2-induced revertants suggest that certain types of DNA damage are more recombinogenic in EM9 than in 'normal' CHO cells, which possibly reflects the specific mutation in the former cell line.

Animals↗
PubMed1989

Analysis of emergency department management of suspected bacterial meningitis.

Previous studies of emergency department management of bacterial meningitis have indicated that there are often long delays before initiation of antibiotics. The purpose of our study was to determine whether these delays were related to specific aspects of patient management. From 1981 through 1988, we retrospectively reviewed the medical records of 122 patients primarily evaluated in the ED and admitted for suspected bacterial meningitis at a university (55) and a community (67) hospital. The median time (interquartile range) from ED registration until initiation of antibiotics (time to antibiotics) was 3.0 hours (1.6 and 4.3 hours, respectively) (total range, 0.5 to 18 hours). The time to antibiotics was not significantly related to the time of ED registration. Ninety percent of the total time to antibiotics occurred after the initial physician encounter. Time to antibiotics was significantly (P less than .00005) longer for patients in whom computed tomography scan and/or laboratory analysis of cerebrospinal fluid preceded initiation of antibiotics compared with patients in whom antibiotic administration was not contingent on the results of these procedures (4.3 [3.2 and 6.0] versus 1.9 [1.2 and 3.4] hours, respectively). Also, time to antibiotics was significantly (P less than .00005) longer for patients in whom antibiotics were initiated on the ward as compared with in the ED (4.5 [3.5 and 6.8] versus 2.2 [1.4 and 3.5] hours, respectively). We conclude that long delays exist in the ED before initiation of antibiotics for cases of suspected bacterial meningitis, and that in general these delays appear to be physician generated and to a great extent potentially avoidable.

Adolescent↗
PubMed1989

The noncovalent complex between DNA and the bifunctional intercalator ditercalinium is a substrate for the UvrABC endonuclease of Escherichia coli.

We have demonstrated that the noncovalent complex formed between DNA and an antitumor bifunctional intercalator, ditercalinium, is recognized in vitro as bulky covalent DNA lesions by the purified Escherichia coli UvrABC endonuclease. It was established that no covalent drug-DNA adduct was formed during the incubation of the drug with DNA or during subsequent incubation with the UvrAB proteins. The nucleoprotein-ditercalinium complexes appear different from those generated by repair of pyrimidine dimers. The UvrA protein is able to form a stable complex with ditercalinium-intercalated DNA in the presence of ATP, whereas both UvrA and UvrB proteins are required to form a stable complex with pyrimidine dimer-containing DNA. The apparent half-life of the UvrA- and UvrAB-ditercalinium-DNA complexes following removal of free ditercalinium is 5 min. However, if the free ditercalinium concentration is maintained to allow the intercalation of one molecule of ditercalinium per 3000 base pairs, the half-life of the UvrA- or UvrAB-ditercalinium-DNA complex is 50 min, comparable to that of the complex of UvrAB proteins formed with pyrimidine dimer-containing DNA. UvrABC endonuclease incises ditercalinium-intercalated DNA as efficiently as pyrimidine dimer-containing DNA. However, unlike repair of pyrimidine dimers, the incision reaction is strongly favored by the supercoiling of the DNA substrate. Because UvrA- or UvrAB-ditercalinium-DNA complexes can be formed with relaxed DNA without leading to a subsequent incision reaction, these apparently dead-end nucleoprotein complexes may become lesions in themselves resulting in the cytotoxicity of ditercalinium. Our results show that binding of excision repair proteins to a noncovalent DNA-ligand complex may lead to cell toxicity.

Antineoplastic Agents↗
PubMed1989

Studies of DNA content in cervical intraepithelial neoplasia by cytologic and histologic flow cytometry.

Flow cytometry was done on 82 specimens of cervical cytologic scrapings and biopsies from patients with cervical intraepithelial neoplasia or condyloma. This study yielded significant results when compared with standard cytologic (p less than 0.01) and histopathologic studies (p less than 0.05). Histologic evaluation by flow cytometry required more examinations and did not give significant results. The authors conclude that cytologic flow cytometry, though not a replacement for standard cytologic examination, may be of help in establishing therapeutic strategies and follow-up protocols for patients with cervical precancerous conditions.

Aneuploidy↗
PubMed1988

Purification and characterization of phospholipase A2 inhibitory proteins from pig thyroid gland.

A 32 kDa phospholipase A2 inhibitory protein was isolated from pig thyroid gland after calcium precipitation and fast protein liquid anion-exchange chromatography. SDS-polyacrylamide gel electrophoresis revealed the purity of the protein. The protein activity was assessed by the inhibition of pancreatic phospholipase A2 on [3H]oleic acid-labelled Escherichia coli membranes as substrate and on the prostaglandin E2 production of cultured thyroid cells. The amino acid composition and the isoelectric point were quite similar to those of endonexin previously described in other tissues or cells. The cross-reactivity of a polyclonal antibody against a 32 kDa lipocortin from human peripheral blood mononuclear cells with our thyroidal 32 kDa protein confirmed its lipocortin nature. Before the purification by fast protein liquid chromatography, the Ca2+ pellet contained lipocortin I (35 kDa and its core protein 33 kDa) identified by its cross-reactivity with a polyclonal antibody.

Amino Acids↗
PubMed1988

Induction of an abortive and futile DNA repair process in E. coli by the antitumor DNA bifunctional intercalator, ditercalinium: role in polA in death induction.

Ditercalinium, an antitumor bifunctional intercalator which forms a high affinity reversible complex with DNA, was found to be specifically cytotoxic for polA and lig7 E. coli strains. In the polA strain, the cytotoxic effect of ditercalinium was suppressed by the uvrA mutation. DNA single strand breaks accumulated in presence of ditercalinium at high temperature in lig7 strains but not in polA strains. Ditercalinium caused no DNA synthesis inhibition although it was able to induce SOS functions. It is proposed that the ditercalinium DNA complex because of its non covalent nature acts as a dummy lesion for the UV repair system in E. coli leading to a futile and abortive repair process. Polymerase I appears to be required to prevent the malfunctioning of a DNA repair process triggered by molecules forming non covalent complex with DNA.

Bacterial Proteins↗
PubMed1988

Activation of adenylate cyclase in rat fat cells promotes an increase in GTP content which controls the enzyme activity.

In previous studies, we demonstrated that the treatment of adipocytes with cholera toxin or Bordetella pertussis toxin (IAP) promoted an increase in the total guanosine triphosphate (GTP) content of the cells concomitant with the increase in cyclic adenosine monophosphate (AMP) level and the resulting lipolysis. In the present studies, we show that the acute challenge of fat cells with 1 microM isoproterenol (IPNE) is associated with a transient increase in GTP level (3-fold in 6 min). This increase may be attributed to an inhibition of the disposal of GTP or to a stimulation of its synthesis. To evaluate the actual role of GTP, we used virazole, an antitumor agent which inhibits inosinic acid dehydrogenase. After 2 h preincubation of the cells with 1 mM virazole, the effect of a 6 min challenge with 1 microM IPNE is decreased by 59% at the GTP level and by 42% in cyclic AMP production. One hour later, the resulting lipolytic efficiency is reduced by 57%. IAP treatment (10 micrograms/ml) produced its maximal effect on GTP and cyclic AMP levels and on lipolysis after 90 min incubation. The antilipolytic effect of 1 microM phenylisopropyladenosine (PIA) is almost abolished. When 1 mM virazole is added to the cell suspension to deplete the guanyl nucleotide pool, the resulting lipolysis due to IAP treatment is decreased by 85%, whereas GTP and cyclic AMP levels were decreased by 80 and 70%, respectively. We can conclude that the cyclic AMP synthesis in intact cells is accompanied by a parallel increase of their GTP content, whether the stimulation results from the activation of Gs or the inhibition of Gi. The reduction of the guanyl nucleotide pool under virazole results in a relatively less important inhibition of lipolysis when Gs is stimulated than when it is Gi.

Adenosine Triphosphate↗
PubMed1988

Asymmetrical bisintercalators as potential antitumor agents.

Ditercalinium and its analogues are dimeric molecules made up of two identical 7H-pyrido[4,3-c]carbazole rings linked by symmetrical linking chains. These dimers elicit antitumor properties through a new mechanism of action. Recently, a relationship was found between their antitumor properties and their cytotoxic effect on the polA Escherichia coli mutant strain, suggesting that 7H-pyrido[4,3-c]carbazole dimers might induce a DNA deformation that could be recognized by the E. coli SOS repair system. Thus, the role of symmetry in ditercalinium analogues for their DNA binding, antitumor properties, and bacterial toxicity is investigated in the present study, by introducing asymmetric parameters in their structures. Dimers were either synthesized with an asymmetrical rigid linking chain or made up of two chemically different chromophores, i.e., acridine and 7H-pyrido[4,3-c]carbazole. The asymmetrical dimers remain able to bisintercalate into DNA with high affinities, but a dramatic loss in their antitumor potency is observed. On the other hand, these asymmetrical dimers are cytotoxic for polA E. coli mutants, like their symmetrical analogues. These results show that the symmetry plays a crucial role for the antitumor potency in the 7H-pyrido[4,3-c]carbazole dimers series.

Animals↗
PubMed1988

Prostaglandin production and contractile response of umbilical arteries in preeclamptic pregnancies with and without intrauterine growth retardation.

Biochemical and biophysical properties of umbilical arteries from normotensive and preeclamptic pregnancies were examined. The production of prostaglandins E and F, 6-keto-PGF1 alpha, and thromboxane B2 by umbilical arteries from normotensive, mildly preeclamptic, and severely preeclamptic pregnancies were measured in incubation media at baseline and after addition of arachidonic acid. The initial baseline values of 6-keto-PGF1 alpha were decreased in the severely preeclamptic patients with intrauterine growth retardation (IUGR) but not in any of the other groups. Addition of arachidonic acid resulted in a significant increase in 6-keto-PGF1 alpha production over initial baseline in all groups except in the severely preeclamptic pregnancies without IUGR. These results suggest a differential defect in the 6-keto-PGF1 alpha metabolic pathway in severely preeclamptic patients with IUGR compared with those without IUGR. The stretch response curve to serotonin was decreased in the severely preeclamptic group with IUGR compared with the control group. The contractile response to individual vasoactive agents (serotonin, prostaglandin F2, norepinephrine, angiotensin II, and arachidonic acid) showed no significant difference between the normotensive and preeclamptic groups.

Angiotensin II↗